Tetrapeptide

ABSTRACT

The peptide X-Arg-Gly-Asp-R-Y wherein X is H or at least one amino acid and Y is OH or at least one amino acid, and R is an amino acid selected from Thr or Cys, or other amino acid, having the same cell-attachment activity as fibronectin and the peptide X-Arg-Gly-Asp-Ser-Y, wherein X and Y, having said activity are disclosed.

This application is a continuation of application Ser. No. 08/458,833now pending, filed Jun. 2, 1995, which is a continuation of applicationSer. No. 07/902,472, filed Jun. 19, 1992 now pending, which is acontinuation of application Ser. No. 07/660,526, filed Feb. 25, 1991 nowabandoned, which is a continuation of application Ser. No. 07/242,713,filed Sept. 9, 1988, issued as U.S. Pat. No. 5,041,380; which is acontinuation of application Ser. No. 06/744,981, filed Jun. 17, 1985,issued as U.S. Patent No. 4,792,525, which is a divisional ofapplication Ser. No. 06/554,821, filed Nov. 22, 1983, issued as U.S.Pat. No. 4,578,079; which is a continuation-in-part of application Ser.No. 06/405,239, filed Aug. 4, 1982, issued as U.S. Pat. No. 4,517,686;which is a continuation-in-part of application Ser. No. 06/433,457 filedNov. 8, 1982, now abandoned; which is continuation-in-part ofapplication Ser. No. 06/518,036, filed Jul. 28, 1983, issued as U.S.Pat. No. 4,589,881.

This invention is directed to polypeptides related to fibronectin andmore particularly to a polypeptide segment of human fibronectin whichinteracts with cell surfaces. In particular, the invention promotes cellattachment to substrates on which the peptide segment is immobilized,and inhibits cell attachment when presented in solubilized form.

BACKGROUND OF THE INVENTION

Fibronectin is a large glycoprotein, about 450 thousand daltons, whichis composed of several apparently independent functional domains.Fibronectin was earlier discovered as a major extracellular matrixprotein, and it was demonstrated that it would interact in vitro withother structural molecules, such as collagen, glycosaminoglycans,proteoglycans, fibrinogen, fibrin, and actin, as well as with cellsurfaces. It was discovered that fibronectin promotes the attachment ofsuspended cells to collagen and also that it promotes the attachment ofsuspended cells directly to tissue culture substrate, independent of itsbinding to collagen. Accordingly, investigation continued with respectto the region of the fibronectin molecule that interacts with cellsurfaces.

Earlier, a polypeptide fragment of fibronectin which embodies the cellattachment activity of fibronectin was isolated, purified andcharacterized as a 11.5 kDal polypeptide of 108 amino acid residues, andhaving the formula:H-Ile-Gly-Gln-Gln-Set-Thr-Val-Ser-Asp-Val-Pro-Arg-Asp-Leu-Glu-Val-Val-Ala-Ala-Thr-Pro-Thr-Ser-Leu-Leu-Ile-Ser-Trp-Asp-Ala-Pro-Ala-Val-Thr-Val-Arg-Tyr-Tyt-Arg-Ile-Thr-Tyr-Gly-Glu-Thr-Gly-Gly-Asn-Ser-Pro-Val-Gln-Glu-Phe-Thr-Val-Pro-Gly-Ser-Lys-Ser-Thr-Ala-Thr-Ile-Ser-Gly-Leu-Lys-Pro-Gly-Val-Asp-Tyr-Thr-Ile-Thr-Val-Tyr-Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro-Ile-Ser-Ile-Asn-Tyr-Arg-Thr-Glu-Ile-Asp-Lys-Pro-Ser-Gln-Met-OH.Also, a fragment of the foregoing molecule having the same cellattachment activity was synthesized and is comprised of 30 amino acidresidues having the formula:H-Tyr-Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro-Ile-Ser-Ile-Asn-Tyr-Arg-Thr-Glu-Ile-Asp-Lys-Pro-Ser-Gln-Met-OH.(These fragments have been described in the aforesaid patentapplications.) These polypeptides, or a fragment thereof which has cellattachment activity, can be used to prepare substrates to which cellswill attach. Such substrates are useful in cell culture dishes and arealso useful for employment in medical prosthetic devices forimplantation in the human body where enhanced cell attachment to thesurface is desirable.

SUMMARY OF THE INVENTION

Here we wish to describe the precise localization of this function ofthe fibronectin molecule as a tetrapeptide sequence. This sequence or achemically similar, biologically equivalent, sequence is shared by someother proteins which interact with cells. These include collagen,fibrinogen and surface proteins of E. coli bacteria and Sindbis virus.These findings suggest that the mechanism by which cells attach to afibronectin-containing substrate may be only one instance of a widelygeneral recognition system that cells use to adhere to any number ofsubstrates. This mechanism may also be involved with a cell's phagocyticactivity. Further, it also appears possible that bacteria and possiblyeven certain viruses may take advantage of this universal cellularadhesion mechanism to gain entry into the body or the cell.

The present invention contemplates a new composition, a polypeptidewhich alters the cell attachment activity of cells to various substratesindependent of its binding to collagen, affects cell phagocytosis, andwhich consists essentially of an isolated tetrapeptideX-Arg-Gly-Asp-Ser-Y wherein X is H or one or more amino acids and Y isOH or one or more amino acids. The tetrapeptide composition ischaracterized in that it is substantially isolated from fibronectin,either by separation from fibronectin or by synthesis whereinfibronectin was never present, and has substantially the same cellattachment activity as fibronectin. In defining the tetrapeptide, thereis some variability in one of the amino acids. While Arg-Gly-Asp-Ser isthe preferred form of the tetrapeptide of this invention, it may includeother amino acids additionally or in a limited sense in substitution forone or more of the amino acids, such as for Ser, Arg-Gly-Asp-Cys orArg-Gly-Asp-Thr which exhibit a similar cell attachment activity.Chemical moieties may be included at either end, typically at the --COOHend, of the, tetrapeptide for the purpose of immobilizing the peptide,or, amino acid additions may be used to modify the cell attachmentactivity. Also, the invention may be incorporated as part of a largermolecule.

The present invention also contemplates the method of using thesecompositions to promote cell attachment to a substrate wherein theinvention is immobilized on the substrate.

The present invention additionally contemplates the method of using theinvention in a solubilized or suspended form to inhibit undesirable cellattachment to a substrate or to each other, and to enhance thephagocytic activity of the cells.

DESCRIPTION OF THE DRAWINGS

FIG. 1 lists the polypeptides, and their respective amino acid sequenceand relative cell attachment activity in concentrations necessary toachieve half-maximal activity, that were synthesized in determining thesmallest peptide exhibiting cell attachment activity.

FIG. 2 compares cell attachment activity of selected synthesizedpolypeptides from FIG. 1 with fibronectin (FN).

FIG. 3 lists the proteins ocurring naturally containing substantiallythe tetrapeptide sequence of amino acids, the fragment sequencesynthesized (in bold type) containing the active site, and the cellattachment activity if tested.

    ______________________________________                                        Abbreviations for Amino Acids                                                                    Three-letter                                                                            One-letter                                         Amino Acid       abbreviation         symbol                                ______________________________________                                        Alanine        Ala       A                                                      Arginine                     Arg               R                              Asparagine                   Asn               N                              Aspartic acid                Asp               D                              Cysteine                     Cys               C                              Glutamine                    Gln               Q                              Glutamic acid                Glu               E                              Glycine                      Gly               G                              Histidine                    His               H                              Isoleucine                   Ile               I                              Leucine                      Leu               L                              Lysine                       Lys               K                              Methionine                   Met    M                                         Phenylalanine                Phe               F                              Proline                      Pro              P                               Serine                       Ser   S                                          Threonine                    Thr              T                               Tryptophan                   Trp              W                               Tyrosine                     Tyr              Y                               Valine                       Val              V                             ______________________________________                                    

he nomenclature used to define the polypeptide is that specified bySchroder & Lubke, "The Peptides", Academic Press (1965) wherein, inaccordance with conventional representation the N-terminus appears tothe left, and the C-terminus appears to the right. Where the amino acidresidue has isomeric forms, it is the L-form of the amino acid that isrepresented.

The Lnvention provides a polypeptide having the following formula:H-Arg-Gly-Asp-Ser-OH and is intended to include other polypeptides orsubstances containing this formula as well as polypeptides formed fromthe invention by limited substitution or deletion and which have cellattachment activity. (Cell attachment activity hereinafter includes cellattachment promoting activity, phagocytic activity, and the inhibitionof cell attachment.) Moreover, the coupling of the peptide to substratesmay be faciliated in certain instances, without affecting the cellattachment activity, by adding a Cys residue at the C-terminus. Further,the cell attachment activity may be modulated by variable additions tothe C- and/or N-termini.

The invention, or a larger polypeptide or other molecule including theinvention, can be synthesized by any suitable method, such as byexclusively solid-phase techniques, by partial solid-phase techniques,by fragment condensation, or by classical solution addition. It is alsoenvisioned that a hybrid protein with adhesive or cell attachmentproperties could be generated by combining the peptide of the inventionwith another protein. Moreover, using recently developed recombinant DNAtechniques, the invention may be synthesized singularly, or combinedwith another protein by first including the DNA code of the invention inthe code of the desired protein.

Source of Peptides. The peptides are preferably prepared using solidphase synthesis, such as that described by Merrifield, J. Am. Chem.Soc., 85, 2149 (1964), although other equivalent chemical synthesesknown in the art, as mentioned above, can also be used. Solid-phasesynthesis is commenced from the C-terminus of the peptide by coupling aprotected alpha-amino acid to a suitable resin, as generally set forthin U.S. Pat. No. 4,244,946, issued Jan. 21, 1982 to Rivier et al., thedisclosure of which is incorporated herein by reference. Examples ofsyntheses of this general type are set forth in U.S. Pat. Nos. 4,305,872and 4,316,891. Discussion of the solid-prase synthesis of a 41-residuepolypeptide is set forth in Science, 213, 1394-1397 (Sept. 1981) in anarticle by Vale et al., which refers to a more detailed discussion ofthe synthesis, which appears in an article by Marki et al. in J. Am.Chem. Soc., 103, 3178 (1981).

In order to locate the cell attachment activity of the fibronectinmolecule which would possibly be contained within a hydrophilic stretchof amino acids in the 30-amino acid synthetic peptide described above, anumber of peptides were synthesized using the Merrifield procedure byPeninsula Laboratories (Belmont, Calif.), and included, in most cases, aCOOH-terminal cystine. The design was to selectively synthesize peptidessmaller than a previous fragment exhibiting cell attachment activity. Ascan be clearly seen in FIG. 1, this approach permits the determinationof the smallest fragment showing activity.

Cell Attachment Promoting Activity. Those peptides having aCOOH-terminal cystine residue were assayed for their activity inpromoting cell attachment by first attaching them via theheterobifunctional crosslinker SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate, Sigma, St. Louis, Mo.) to rabbit IgG which was immobilizedon plastic. The attachment assay was then carried out as described byRuoslahti, E. et al., Methods Enzymology 82: 803-831 (1981) usingfreshly trypsinized normal rat kidney cells (NRK). After a one hourincubation, those cells which had attached were fixed, stained andquantitated using either an Artek cell counter or a multiscanspectrophotometer. In all cases, maximum attachment was about 80-90% ofthe cells plated. The relative activity of each peptide is indicated bythe concentration of the peptides in nmoles/ml necessary to achievehalf-maximal activity, or, where accurate quantitation was not possible,by the crosses at the right of FIG. 1, and by the graph in FIG. 2.

Among the peptides tested, the tetrapeptide Arg-Gly-Asp-Ser was thesmallest one and the only tetrapeptide which, when properly immobilized,had the property of causing cell attachment. The activity of thepeptides decreased somewhat with size (FIG. 2). This decrease may be dueto a decrease in the stability of their conformation or to a relativeinaccessibility on the substrate. The serine residue could be replacedby a cysteine residue without complete loss of the activity of thepeptide. When the arginine or aspartic acid residues were selectivelydeleted, however, the activity was lost. Moreover, substitution of theglycine with the bulky valine residue also abolished the activity(RVDSPAC, FIG. 1). Whereas these results show that the residues criticalfor the activity reside in the tetrapeptide sequence, amino acidsimmediately flanking the four residue fragment may have a modulatoryeffect on their activity.

It appears that while the tetrapeptide described is the determinant,optimum size of the polypeptide is about a hexapeptide which includesthe defined tetrapeptide. Thus, the invention consists essentially ofthe tetrapeptide which would preferrably be part of a hexapeptide.

Arg-Gly-Asp-Ser and Related Sequences in Other Proteins. Havingestablished the importance of the tetrapeptide sequence, a computersearch through the published protein sequences was conducted by theNational Biomedical Research Foundation (George Washington UniversityMedical Center, Washington, D.C.). Also included were sequences in whichthe serine was allowed to be replaced by other amino acids, and thearginine and aspartic acid by the chemically similar lysine and glutamicacid, respectively. The search through other proteins revealed fiveproteins having the Arg-Gly-Asp-Ser sequence (FIG. 3).

Of the five proteins having the identical four amino acid sequence,fibrin(ogen) is of most obvious interest because of its demonstratedinteraction with fibronectin and cell surfaces. For this reason, asynthetic nonopeptide designed after the fibrin sequence was tested. Ascan be seen in FIG. 3, it too was an active cell attachment promoter.Attempts to demonstrate the attachment of test cells to intactfibrinogen or fibrin have giver, no clear-cut results, althoughplatelets do bind to the fibrinogen molecule.

Among the remaining proteins that possess sequences related to theactive tetrapeptide, the family of collagenous proteins is of particularinterest for two reasons. First, collagens have been shown to mediatecell attachment independently of fibronectin, and, secondly, variationsof the tetrapeptide sequence are particularly abundant in collagens.These sequences are repeated at fairly regular intervals along the α₁and α₂ chains of type I collagen.

Four synthetic peptides were prepared based on diverse sequences incollagen. In three of these, the serine residue was replaced bythreonine, hydroxyproline, or lysine, respectively. In the fourth one alysine was substituted for arginine and glutamic acid for aspartic acid.Of these peptides only the serine to threonine substitution was activewith the NRK test cells (FIG. 2). Other chemically similar substitutionssuch as serine to alanine may also yield active sites.

The results show that the primary cell-binding site of the fibronectinmolecule resides in an extremely short amino acid sequence which isshared by at least one other adhesive protein, collagen. Because of thenature of the proteins having the tetrapeptide sequence among theproteins searched, the results suggest that the tetrapeptide withselected substitutions, may represent a universal attachment mechanism.

Inhibition of Cell Attachment. If the mechanism of cell attachmentinvolves the recognition of the amino acid sequence of the tetrapeptideby a receptor on the cell, then it could be postulated that attachmentcould be inhibited by preventing this recognition by "blockings" thereceptor. To demonstrate this inhibitory function of the invention,fibronectin was immobilized on a substrate to be tested for cellattachment activity. In separate experiments, various concentrations ofthe tetrapeptide (Arg-Gly-Asp-Ser) and a hexapeptide,Pro-Arg-Gly-Asp-Ser-Gly in a solubilized form were combined with thefree cells, and attachment activity measured as above. Both peptideswere shown to inhibit the normal attachment of cells to afibronectin-coated substrate when placed in a solubilized form incombination with the free cells. The concentration necessary to exhibithalf-maximal cell inhibition activity was 0.6-0.8 mmoles/ml and 0.3mmoles/ml for the tetrapeptide and the hexapeptide, respectively.

Enhanced Phagocytic Activity. Fibronectin has been shown to promotephagocytosis, and this activity has been linked to the cell attachmentactivity. An application that can be envisioned for the invention basedon these observations is to promote the entrance to cells of particlescontaining, for example, a therapeutic agent, by administering theparticles with the invention in a solubilized form.

Practical application such as the preparation of surfaces for optimalcell culture, the derivatization of various prosthetic materials topromote bonding with surrounding tissues, a method to provide for theincreased internalization of molecules such as toxins, drugs, hormones,or the like by the enhancement of phagocytosis, and the development ofways of manipulating cellular adhesion mechanisms in diseases such ascancer metastasis and platelet aggregation can also be envisioned. Sincea peptide of four amino acids is unlikely to have more than one bindingsite, one question that can be addressed now is whether the interactionof all types of cells with fibronectin involves this same region of themolecule. Platelets, for example, may bind fibronectin on their surfacesby a different mechanism. This would be important in using this peptideto regulate cell attachment or in the design of prosthetic materials. Itwould also shed light on the role played by fibronectin in vivo.

In particular, the coating of the culture substrate with thecell-attachment polypeptide obviates the use of fibronectin in themedium, thus providing better defined conditions for the culture as wellas better reproducibility. As one example of commercial use ofcell-attachment surfaces, Cytodex particles, manufactured by Pharmacia,are coated with gelatin, making it possible to grow the same number ofadherent cells in a much smaller volume of medium than would be possiblein dishes. The activity of these beads is generally dependent upon theuse of fibronectin in the growth medium, and the cell-attachmentpolypeptide is expected to provide an improved, chemically-definedcoating for such purposes. Other surfaces or materials may be coated toenhance attachment, such as glass, agarose, synthetic resins, orlong-chain polysaccharides.

Medical devices can be designed making use of such substrata to attractcells to the surface in vivo or even to promote the growing of a desiredcell type on a particular surface prior to grafting. An example of suchan approach is the induction of endothelial cell growth on a prostheticblood vessel or vascular graft, which is generally woven or knitted fromnitrocellulose or polyester fiber, particularly Dacron (polyethyleneterephthalate) fiber. Most types of cells are attracted to fibronectinand to this polypeptide, but endothelial cells and fibroblastic cells inparticular are attracted to fibronectin. The latter point indicates thepotential usefulness of this defined polypeptide in coating a patchgraft or the like for aiding wound closure and healing following anaccident or surgery. In such cases, it may be advantageous to couple thepeptide to a biological molecule, such as collagen, a glycosaminoglycanor a proteoglycan; for example, the five-residue fragment having aCys-residue at the C-terminus coupled to monomeric collagen by using acrosslinker such as 3-(2-pyridyldithilo) propionic acidN-hydroxysuccinimide ester to effect cross-linking of the cysteine to alysine residue in the collagen, or by using carbodiimide without the Cyson the four-residue fragment. It is also indicative of its value incoating surfaces of a prosthetic device which is intended to serve as atemporary or semipermanent entry into the body, e.g. into a blood vesselor into the peritoneal cavity, sometimes referred to as a percutaneousdevice. Fibronectin has been shown to be chemotactic to fibroblasts andmacrophages. This activity correlates with the presence of the cellattachment domain. One potential manifestation of the cell attachmentactivity of the synthetic peptides described here, and fragments thereofof like characteristics, is chemotactic activity.

Application of the ability of the invention to inhibit cell attachmentwhen presented in a solubilized form may find utility in situationswhere it is desirable to prevent cell adhesion to a substrate oradhesion between cells. Undesirable cell attachment to various medicalsutures, or dressings, may be prevented by presenting the invention insolubilized form. When the invention is used either in conjunction orcombination with another molecule, such as a therapeutic agent, orparticle treated with such an agent, the entrance of that agent orparticle into the cell may be enhanced by the effect of the invention onthe phagocytic activity of the cell, thereby increasing the efficiencyof the therapeutic agent.

Although the invention has been described with regard to certainpreferred embodiments, it should be understood that various changes andmodifications, as would be obvious to one having the ordinary skill inthis art, may be made without departing from the scope of the inventionwhich is defined in the appended claims. For example, it may not benecessary to have the free acid at the C-terminus, as it may be amidatedor substituted by some other group. Further, limited substitutions maybe made to the basic tetrapeptide as illustrated by the substitution ofSer by Cys, without destroying the innerent cell attachment activity.Particular features of the invention are emphasized in the claims whichfollow.

Industrial Application

The invention is useful in surgery and therapeutic reconstruction andtreatment of injuries.

What is claimed is:
 1. A hybrid protein having cell adhesive properties,comprising a peptide of the following formula:

    X-Arg-Gly-Asp-R-Y

wherein X is H or one or more amino acids, Y is OH or one or more aminoacids, and R is an amino acid selected from Ser, Thr, Cys, or otheramino acid, and said peptide has about 31 amino acids or less, has cellattachment activity and is combined with a non-adhesive protein moleculeto form a hybrid protein having cell adhesive properties.
 2. Ahybridprotein of claim 1, wherein the peptide has fifteen amino acids or less.3. A hybrid protein of claim 2, wherein the peptide has ten amino acidsor less.
 4. A hybrid protein of claim 3, wherein the peptide has sixamino acids or less.
 5. A composition of matter comprising a peptide ofthe following formula:

    X-Arg-Gly-Asp-R-Y

wherein X is H or one or more amino acids, Y is OH or one or more aminoacids, and R is an aminc acid selected from Ser, Thr, Cys, or otheramino acids, and the peptide has about 31 amino acids or less, has cellattachment activity and is coupled with a biological molecule selectedfrom the group consisting of a collagen, glycosaminoglycan, or aproteoglycan.
 6. The composition of matter of claim 5 produced bychemical means employing a cross-linker.
 7. The composition of matter ofclaim 6, wherein the biological molecule is collagen.
 8. The compositionof matter of claim 7, wherein the cross-linker is 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide.
 9. A composition of matter of claim5, wherein the peptide has fifteen amino acids or less.
 10. Acomposition of matter of claim 9, wherein the peptide has ten aminoacids or less.
 11. A composition of matter of claim 10, wherein thepeptide has six amino acids or less.